ABSTRACT Whether apoptotic stimuli arise from the nucleus, cell membrane surface, or the mitochondria, ultimately, the stimuli converge on a process of activation of a family of cysteine proteases known as the caspases (cysteine aspartases). Activation of caspase family members mediate programmed cell death in normal physiology and a number of diseases, with caspase-3 standing at the center of the cell death program. Existing in a non-active pro-enzyme form in the cytosol of resting cells, caspase-3 is one key effector protease when activated. Thus, to monitor the final commitment of cells to death pathways, the need exists to directly quantify the enzymatic activity of caspase-3 in vivo. To meet this challenge, we designed and synthesized a class of peptide-based imaging agents that can penetrate the cell via non-receptor-mediated endocytic pathways, gaining access to the cytosolic compartment. These cell- penetrating optical imaging agents contain quenched fluorophores flanking target protease sequences which are cleaved and activated by caspase-3. Upon cleavage, these agents show caspase-3- dependent fluorescence signal amplification, thereby enabling high quality enzyme-specific molecular imaging of intracellular processes in vivo. Importantly, we discovered that upon intravitreal (intraocular) administration, our cell-penetrating peptides selectively accumulate in retinal ganglion cells (RGCs), the retinal neurons which are selectively injured and degenerate in glaucoma. RGCs are particularly accessible through an intravitreal approach, a routine ophthalmological procedure now performed everyday in the clinic. Thus, we plan to exploit this unanticipated and exciting cell-type-specific property of our peptides in retinal pathology for molecular imaging applications in ophthalmology. This renewal application is focused on translation of this strategy through advanced pre-clinical studies and development of a lead peptide. We propose pre-clinical testing in glaucoma models, toxicology testing, and metabolite profiling. We will advance a lead peptide into the clinic through pilot studies in a unique non-human primate model of glaucoma. These activities benefit from the combination expertise of this applicant team in chemistry, molecular imaging, biochemistry, and vision biology.